Method and compositions for treating injury

ABSTRACT

D-factor, growth hormone, IL-1, and tumor necrosis factors are employed alone or in combination as synergistic cytoprotective agents for patients exposed to toxic doses of ionizing radiation and/or chemotherapy. Synergistic cytoprotective regimens are provided. Use of D-factor, growth hormone, tumor necrosis factors, and/or IL-1, alone or in synergistic combination, is disclosed for the prevention and treatment of alopecia.

This is a divisional of applications Ser. No. 08/076,086 filed on Jun.10, 1993, which is a continuation application of Ser. No. 07,602,849filed on Oct. 26, 1990, (now abandoned) which is a continuation-in-partapplication of Ser. No. 07/507,341 filed on Apr. 10, 1990, (nowabandoned) which applications are incorporated herein by reference.

This invention relates to the treatment of injury, especially injuryresulting from radiation or chemotherapy. In particular, it relates tothe amelioration of injury to otherwise healthy tissue concomitant withthe treatment of cancers and other proliferative disorders, and in thecourse of bone marrow transplants.

Alopecia is the loss or absence of hair from skin areas where itnormally is present; it encompasses male pattern baldness, inflammatoryhair loss, drug-induced hair loss, radiation-induced hair loss, and avariety of syndromes. It has recently been reported that IMUVERT, abiologic response modifier derived from the bacterium Serratiamarcescens, protected against alopecia in 8-day-old rats treated withcytosine arabinoside and doxorubicin, but not against alopecia inducedby cyclophosphamide, Hussein et al., Science 249:1564-1566 (28 Sept.1990).

D-Factor (hereafter "DF") is a known molecule. It is capable ofdirecting the choice of neurotransmitter phenotype made by cultured ratsympathetic neurons, regulates the growth and differentiation ofembryonic stem cells and myeloid cells and stimulates bone remodelingand acute-phase protein synthesis in hepatocytes. It has been termedDIA, DIF, DRF, HSFIII, human interleukin DA (HILDA) and LIF (leukemiainhibitory factor). This cytokine has been compared to IL-6 and TGF-betain that it regulates function, growth and differentiation in the embryoand in the adult in many tissues and cell types, including monocyticcells, megakaryocytes, embryonal stem cells, hepatocytes, adipocytes,osteoblasts and neuronal cells. See Yamamori et al., "Science" 246:1412(1989), Lowe et al., "DNA" 8(5):351 (1989) and Abe et al., "J. Biol.Chem." 264(15):8941 (1989). DF also is known to induce differentiationof myeloid leukemia cells and therefore has been proposed in the art tobe useful in the treatment of myeloid leukemias. DF is currentlypurified by complex methods not amenable to large scale commercialdevelopment (Hilton et al., "Analyt. Biochem." 173:359 1988!).

Human growth hormone (hGH) is secreted in the human pituitary. Itsmature form consists of 191 amino acids and has a molecular weight ofabout 22,000; its sequence and characteristics are set forth, forexample, in Hormone Drugs, Geuriguian et al., U.S.P. Convention,Rockville Md. (1982) incorporated herein by reference.

hGH has been used for the treatment of hypopituitary dwarfism, and hasbeen proposed for the treatment of burns, wound healing, dystrophy, boneknitting, diffuse gastric bleeding and pseudarthrosis. The majorbiological effect of hGH is to promote growth. The organ systemsaffected include the skeleton, connective tissue, muscles, and viscerasuch as liver, intestine, and kidneys. Growth hormone exerts its actionthrough interaction with specific receptors on cell membranes.Administration of growth hormone for treatment of pulmonary dysfunctionand ventilator dependency has been proposed in U.S. Ser. No. 07/306,978filed 7 Feb. 1989.

Interleukin-1 (IL-1) is produced by activated macrophages. At least twotypes exist, designated α and β (March et al., Nature 315:641-647(1985)). IL-1 mediates a wide range of biological activities; it hasbeen found to stimulate fibroblast proliferation and to induce in thesecells the synthesis of collagenase, prostaglandin E₂ and interferonbeta-2, to decrease in adipocytes the activity of lipoprotein lipase,and to activate osteoclasts.

Tumor necrosis factors (TNFs) are polypeptides produced bymitogen-stimulated macrophages (TNF-α) or lymphocytes (TNF-β) which arecytotoxic to certain malignantly transformed cells but not to certainnormal cells (E. A. Carswell et al., Proc. Natl. Acad. Sci. U.S.A.72:3666, 1975; B. J. Sugarman et al., Science 230:943, 1985; Schultze etal. , J. Immunol. 140: 3000, 1988).

TNF-α has been suggested to be responsible for wasting and cachexia inpatients with cancer or severe infections, and both TNF-α and TNF-βmediate many other biological effects. TNF is also known to induce MHCantigens. It has been reported that TNF induces MnSOD in varioustransformed and normal cell lines (Wong et al., Science 242:941, 1988).

It has been suggested repeatedly in the literature that TNF at certaindosages enhances tissue injury caused by reactive oxygen species (see,e.g. Clark et al., J. Cell Biochem. Suppl. 12A, p. 40, January 1988;Sullivan et al., Infect. and Immunity 56(7): 1722-1729, 1988; and Tiegset al., Biochem. Pharmacol. 38(4): 627-631, 1989).

The literature has reported that TNF-α and other cytokines such as IL-1may protect against the deleterious effects of ionizing radiationproduced during a course of radiotherapy, such as denaturation ofenzymes, lipid peroxidation, and DNA damage (Neta et al., J. Immunol.136(7):2483, 1987; Neta et al., Lymphokine Res. 5:s105, 1986; Neta etal., Fed. Proc. 46:1200 (abstract), 1987; Urbaschek et al. , LymphokineRes. 6:179, 1987; U.S. Pat. No. 4,861,587; Neta et al., J. Immunol.140:108, 1988), and that TNF treatment accelerates restoration ofhematopoiesis in animals compromised by sublethal doses of cytotoxicdrugs or irradiation (Neta, et al., Blood 72(3):1093, 1988). It has beenreported that pretreatment with TNF protects mice from lethal bacterialinfection (Cross et al., J. Exp. Med. 169:2021-2027, 1989). It has alsobeen suggested that administration of subdeleterious amounts of TNFand/or IL-1 may modulate the deleterious effect of subsequent TNF and/orIL-1 administration; this reference further suggests that ionizingradiation may be administered as a sensitizing agent (EPO Appl. EP 0 259863 A2). It has also been reported that pretreatment of cells witheither TNF or IL-1 can confer resistance to killing by subsequenttreatment with TNF-α and cycloheximide in combination (Wallach, J.Immunol. 132:2464, 1984; Hahn et al., Proc. Natl. Acad. Sci. U.S.A.82:3814, 1985; Holtamann et al., J. Immunol 139:1161, 1987; Wong et al.,Cell 58:923, 1989). It has been suggested that inadequate endogenouslevels of TNF may be involved in the development of diabetes and oflupus erythematosus (Jacob et al., Nature 331:356-358, 1988).

Ionizing radiation damages tissue in large measure by generating oxygenfree radicals which in turn readily react with the neighboring tissuecomponents such as nucleic acids and proteins. However, ionizingradiation damages some tissues differently than others. In particular,rapidly proliferating cells such as those of bone marrow are speciallysensitive to ionizing radiation, while tissues made up of mature,differentiatal cells are most likely to survive doses of ionizingradiation. This differential effect has been used to treat cancers,which as rapidly proliferating cells are more sensitive to radiationthan non-neoplastic cells, and in preparing patients for bone marrowtransplantation.

Allogenic bone marrow transplantation typically is used to treatleukemias. In this procedure the patient is treated withchemotherapeutic agents such as cyclophosphamide and with whole bodyirradiation in order to completely kill the patient's own cancerous bonemarrow, after which the bone marrow transplant (from an autologous donoror remission marrow from the patient) is injected into the patient andultimately lodges in the marrow. Even in successful therapies where thetransplant becomes functional, the patient is seriously affected by sideeffects of the therapy such as infections, radiation damage tonon-target tissues, and alopecia. Radiation therapies for other cancersare also fraught with marty of the same adverse reactions, particularlyinfections and a general weakening and debilitation of patients who areotherwise in no condition to tolerate such injury.

Thus, considerable effort has been expended by the clinical community inattempts to stem radiation injury without also dispensing with thedesired therapeutic effects. This effort has been directed at focusingthe radiation on the tumors, by attempting to decrease the hypoxiccondition in which many solid tumors are found (for example, byhyperbaric oxygen breathing), and by administering cytoprotectivesubstances.

A number of substances have been advocated as cytoprotectors, and somehave received clinical use. According to Rubin et al., "ClinicalOncology" p. 70 (1983), sulfhydryl containing compounds such as cysteineand cysteamine have long been known to be radiation protective. It isbelieved that these sulfhydryl compounds act as free-radical scavengersby entering into the cell and there protecting from radiation damage byreducing the yield of damaging free radicals. Some cytoprotectors arepostulated to work by being taken up by normal cells but not inappreciable amounts by target cells such as neoplasms. See Yuhas et al.,"J. Natl. Cancer Inst." 42:331 (1969) and Fletcher, "Textbook ofRadiotherapy" (1980). Recently, it has been observed that TNF is capableof radioprotection of normal tissues in HIV therapy, but only whenadministered prior to exposure to radiation (U.S. Ser. No. 07/417,993,filed Oct. 6, 1989 and now pending).

The principal deficiency shared by known cytoprotective agents usedclinically to date for this purpose is that they are relativelyineffective in reducing mortality and morbidity after exposure toradiation. This would appear to be consistent with the theory that theadverse clinical effects of radiation are merely the manifestation ofcumulative cellular injury caused by highly reactive, transitoryfree-radical intermediates generated at the time of radiation exposure.Based on this theory, one would expect that post-exposure treatmentwould be ineffectual since the injury would already have been sustainedby the cells. This is particularly the case with cytoprotective agentssuch as proteins that are not small molecules and therefore must act byinitiating intracellular events or by active transport into the cell, orboth. The cell's ability to perform these protection-initiatingfunctions would be problematic after a damaging dose of ionizingradiation. Nonetheless, treatment strategies involving a highlyeffective post-exposure cytoprotective agent would be extremely usefulin the therapeutic setting since often times it is difficult to predictthe level of radiation dosing that will be safe and effective for anygiven patient. In addition, such a post-exposure cytoprotective agentwould be useful in the event of accidental or unexpected radiationexposure to health workers or others.

Accordingly, it is an object of this invention to provide cytoprotectiveagents and treatment strategies that are highly effective after exposureto radiation.

It is another object of this invention to provide methods and agents forreducing cellular damage in chemotherapeutic and therapeutic treatments,particularly those employed for HIV infections and neoplasms.

Yet another object of this invention is to provide an agent forprevention or treatment of alopecia.

These and other objects of the invention will be apparent from furtherconsideration of the disclosure.

SUMMARY OF THE INVENTION

Some of the objects of the invention are achieved in a method comprisingexposing a subject to a radiation or chemotherapy injury, theimprovement comprising administering a cytoprotective dose of an agentor agents selected from the group consisting of DF, and/or GH, alone orin combination with TNF or IL-1, to the subject.

Surprisingly, it was found that DF administered to mice 1 hour after theanimals had received a lethal dose of gamma radiation could completelyprotect the animals from death, and that the effect was related to thedose of DF administered. It has been further found that administrationof TNF prior to or concurrent with radiation, followed by administrationof DF or GH after exposure to radiation, provided synergistic levels ofprotection, and that treatment with DF, GH, TNF and/or IL-1, alone or incombination, prevented alopecia associated with radiation andchemotherapy.

This unexpected effect of these agents therefore suggests their use forthe treatment of alopecia, in the therapy of tumors in concert withchemotherapy or radiation. In certain situations, it is preferred toinclude at least one DF or GH administration after the patient isirradiated or exposed to chemotherapy. DF, GH, TNF, and IL-1 may beadministered in a course of treatment excluding chemotherapy orradiation, or prior to and/or subsequenct to chemotherapy orirradiation, together with other agents and therapies heretoforeemployed with chemotherapy or irradiation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the 20 day survived of irradiated mice treated with 7.5micrograms of DF and 1 microgram of DF one hour after irradiation.

FIG. 2 depicts the mature amino acid sequences for human (H) (SequenceID No. 1) and murine (M) (Sequence ID No. 2) D-factor. Areas of homologyare boxed.

FIG. 3 depicts DF's proctective effects on neutrophil recovery inchemotherapeutically treated mice.

DETAILED DESCRIPTION OF THE INVENTION

Tumor necrosis factor or TNF, as employed herein, refers in general tothe various forms of TNF which exhibit one or more biologic propertiesof tumor necrosis such as tumor cell lysis, inhibition of infectiousagents, MHC antigen induction, and neutralization by antibody to TNF-αor TNF-β but not by antibodies to other cytokines. It is believed thatgamma interferon is synergistic with TNF in anti-tumor or anti-viralassays for TNF, and may therefore be desirably administered along withTNF in the practice of this invention.

In particular, the tumor necrosis factors useful herein include TNF-αand TNF-β. The former is described in copending EPO Appl. EP 0 168 214A2 together with methods for its synthesis in recombinant cell culture.Similarly, the latter (previously called lymphotoxin) and suitablerecombinant synthesis methods are described in copending EPO Appl. EP 0164 965 A2. The TNF-α and TNF-β described in these applications includecytotoxic amino acid sequence and glycosylation variants which also areused herein. Of course, TNF-α or TNF-β from non-recombinant sources arealso useful in the practice of this invention.

As used herein, the terms "growth hormone" or "GH" denote growth hormoneproduced from natured source extraction and purification, as well as byrecombinant cell culture systems. "hGH" refers to human growth hormone.See, for example, U.S. Pat. No. 4,321,832, specifically incorporated byreference. The terms likewise cover biologically active human growthhormone equivalents, e.g., those differing by one or more amino acids(s)in the overall sequence. Further, the terms as used in this applicationare intended to cover substitution, deletion and insertion amino acidvariants of hGH, or post-translational modifications.

As used herein, "IL-1" denotes interleukin-1 produced from naturalsource extraction and purification, as well as by recombinant cellculture systems. See, for example, March et al., Nature 315:641-646(1985)), specifically incorporated by reference. The terms likewisecover biologically active IL-1 equivalents, e.g., those differing by oneor more amino acids(s) in the overall sequence. Further, the terms asused in this application are intended to cover substitution, deletionand insertion amino acid variants of IL-1, or post-translationalmodifications.

The mature amino acid sequences for human (H) and murine (M) DF shown inFIG. 2 are disclosed in EP 285,448, published 5 Oct. 1988 (specificallyincorporated by reference herein), especially at FIG. 26, includingmethods for its production in recombinant cell culture. See also D. P.Gearing et al., Nucleic Acids Res. 16:9857 (1988), and N. M. Gough etal., Proc. Nat. Acad. Sci. U.S.A. 85:2623-2627 (1988). For purposes ofthis application, DF is defined herein as any polypeptide having (a)cytoprotective activity as defined below and (b) amino acid sequencewhich is homologous to either amino acid sequence of FIG. 2.

Homologous, as to DF for example, for the purposes herein means that thecandidate polypeptide comprises the epitope of DF that is functional inconferring cytoprotection on experimented animals, or functionallyconserved amino acid variants thereof. Identification of this epitope isa matter of routine experimentation. Most typically, one would conductsystematic substitutional mutagenesis of the DF molecule while observingfor reductions or elimination of cytoprotective activity. This iscommonly accomplished by sequentially substituting each residue of thenative DF sequence with alanine. Preferably, the residues to besubstituted will be those which are (1) identically conserved amonganimal species, (2) located on disulfide bonded loops, and (3)hydrophilic in nature. Domains containing more than 4 identicallyconserved residues among animal species and which are not found withinabout 3 residues of a site of glycosylation are to be preferred formutagenesis. Preferred regions for mutagenesis are 12-25, 34-60 and121-143 of the FIG. 2 sequences. In any case, it will be appreciatedthat due to the size of DF most substitutions will have no effect on DFactivity, but if some effect is seen it will be modestly agonistic orantagonistic. The great majority of DF variants will possess at leastsome cytoprotective activity, particularly if the substitution isconservative. Conservative substitutions are substitutions from the sameclasses, defined as acidic (Asp, Glu), hydroxy-like (Cys, Ser, Thr),amides (Asn, GIn), basic (His, Lys, Arg), aliphatic-like (Met, Ile, Leu,Val, Gly, Ala, Pro), and aromatic (Phe, Tyr, Trp). The 9 C-terminalhydrophobic residues should only be substituted, if at all, with otherhydrophobic residues. The first 3 N-terminal residues may be deleted orfreely substituted, and may include a serine residue inserted at theN-terminus.

Alternatively, or in addition, the active site is identified by raisingantibodies against the intact native DF, screening for neutralizingantibodies, and determining the site to which the neutralizingantibodies bind. Neutralizing antibodies against DF also would find usein diagnostic immunoassays for DF, especially when used in asandwich-type immunoassay in concert with a non-neutralizing antibodydirected against another DF epitope.

Homologous sequences generally will be greater than about 30 percenthomologous on an identical amino acid basis, ignoring for the purposesof determining homology any insertions or deletions from the candidatemolecule in relation to either native sequence of FIG. 2. Homologies ofabout 50, 70 and 90% are also included within the scope hereof so longas the molecules possess the requisite cytoprotective activity.

The cytoprotective agents discussed herein, e.g. DF, hGH, TNF, IL-1,also includes glycosylation variants as well as unglycosylated forms ofthe agents, fusions of the agents with heterologous polypeptides, andfragments of the agents, again so long as the variants possess therequisite cytoprotective activity.

Cytoprotective activity is defined as the statistically significant (Pgreater than 0.05) ability of an intraperitoneal dose of the candidatecytoprotective agent DF and/or hGH, delivered concurrent with or up toabout twenty-four hours after irradiation or chemotherapy, to reducemortality or morbidity of the subject animals. However, it will beunderstood that in therapeutic use it is unlikely, barring accidentaloverdose, that DF would be employed to ameliorate a lethal dose ofradiation. Instead, for example, the typical doses for different humantumors range from 2,000 rad in 2 weeks to 8,000 rad in 8 weeks, with thevast majority of tumors receiving doses between 4,500 and 7,500 rad.These doses are typical therapeutic doses. While they far exceed anyambient radiation they generally are not lethal when administeredproperly over time, but they are characterized by minor to severe sideeffects which can lead to death. DF and/or hGH, alone or in conjunctionwith pretreatment with TNF and/or IL-1, is used either to increase thenet irradiation or chemotherapy dose, with clinical tolerance ofessentially the same toxicity but an advantage in therapy, or to useconventional doses but with amelioration of morbidity and mortality.

Chemotherapy is defined to be the administration of an agent known toexert a toxic effect on rapidly proliferating cells in comparison todifferentiated, mature and slower growing cells. Such agents are wellknown in the therapy of cancers and other proliferative disorders. Theyinclude, for example, adriamycin, chlorambucil, daunomycin,cyclophosphamide, methotrexate, cisplatin, procarbazine, proteinsynthesis inhibiting agents such as actinomycin-D, bleomycin,5-azacytidine, vincristine, alkylating agents, dicarbazine,fluorouracil, hydroxyurea, mitomycin-C, nitrosoureas, busulfan,mithramycin, vinblastine, vindesine, and antimetabolites such asmitrosoureas, cytarabine, and mercaptopurine. These cytotoxic agentscause nausea and vomiting, fever, alopecia, chills and malaise andfatigue. Treatment with a cytoprotective regimen as provided herein (DFand/or hGH, alone or in conjunction with treatment with TNF and/or IL-1)reduces the toxicity of such cytotoxic agents and therefore is useful ascompanion treatment, before and/or after a regimen of chemotherapy.

The amount of DF, GH, TNF and/or IL-1 which is used will depend upon theirradiation or chemotherapy protocol, the condition of the patient, theactivity of the agent used, the administration route, and otherinfluences that will be appreciated by the ordinary artisan. In mice, adosage of about 7.5 micrograms of DF ip/mouse is acceptable, with about0.5 micrograms to 20 micrograms per day being the range from the lowestlevel that is marginally effective for cytoprotection to a safe andadequate upper limit. Obviously, the dose will differ for other animalsand humans and will vary depending upon administration routes.Pretreatment effective amounts of IL-1 administered to mice rangebetween about 0.5 to 25 nanograms per gram of body weight of mouse.

For hGH, a suitable dosage for human administration ranges from 0.001 mgper kg of bodyweight per day to about 0.2 mg per kg of bodyweight perday. Generally, daily dosages of hGH will be from about 0.05 mg per kgof bodyweight per day. Normally, from 0.07 to 0.15 mg/kg, in one or moreapplications per day, is effective to obtain the desired result. In analternative approach, the hGH, particularly where formulated in atimed-release form, may be administered less frequently, i.e., everyother day or every third day for certain indication, such as alopecia.It is presently preferred the hGH be administered within 0 to 24 hoursfollowing exposure to chemotherapy or irradiation.

The therapeutically effective dosage of TNF to be administered to ahuman patient or human tissue generally will range from about 1-250μg/m² per dose, and preferably from about 1-10 μg/m², and mostpreferably 10 μg/m², although the dose of the TNF (as with the othercytoprotective agents) administered will be dependent upon the speciesof the patient, the properties of the TNF employed, e.g. its activityand biological half-life, the concentration of the TNF in theformulation, the rate of dosage, the clinical tolerance of the patientsinvolved, the pathological condition of the patients and the like, as iswell within the skill of the physician. It will be appreciated that thepractitioner will adjust the therapeutic dose in line with clinicalexperience for any given TNF. Preferably, the TNF is administeredintravenously or intramuscularly.

Similar dosage ranges and regimens apply for the treatment of alopecianot associated with radiotherapy or chemotherapy.

The DF, hGH, TNF and IL-1 should be of pharmaceutical grade, i.e.,greater than about 95% pure by weight of protein. It is formulated intoconventional carriers such as isotonic saline at a concentrationsuitable for delivery of a cytoprotective dose in a reasonable volume ofcarrier, typically ranging from several mls. for bolus injection tosevered hundred mls. for administration by iv infusion.

In accordance with this invention, DF, hGH, TNF and/or IL-1 is usedtogether with radiation and/or chemotherapy in the treatment ofleukemias as well as other cancers known to be susceptible to radiationtreatment (e.g., lymphoma, seminoma, dysgerminoma, squamous cell cancerof the oropharyngeal, glottis, bladder, skin and cervical epithelia,adenocarcinomas of the alimentary tract, and astrocytomas) andchemotherapy (e.g. ALL, Wilm's tumor, Ewings sarcoma, retinoblastoma,rhabdomyosarcoma, Hodgkin's disease, DHL, Burkitt's lymphoma, testicularcarcinoma, choriocarcinoma, neuroblastoma, acute leukemia, myeloma,lymphomas, small cell lung cancer, breast, stomach, or adrenal cancer,malignant insulinoma, osteogenic sarcoma, CLL, CGL, prostate,endometrial or cervical carcinoma, endocrine gland carcinomas, malignantcarcinoids, sarcomas, brain cancers, non-oat cell carcinoma, andcarcinoma of the head and neck, thyroid, colon, rectum, liver andbladder.

While it was known to use individually some of the cytoprotective agentsdescribed herein with radiation or chemotherapy, or has been proposed tobe useful in the treatment of myeloid leukemias, the combination ofagents claimed herein unexpectedly and surprisingly offers thesignificant advantage of synergistic and greatly enhanced protectionagainst radiation and chemotherapy toxicity as well. In addition, thecytoprotective agents and regimens claimed herein find use in accordancewith this invention, and the same advantages are obtained, in theradiotherapy or chemotherapy of non-myeloid leukemic cancers, innon-therapeutic (e.g. accidental) irradiation, in bone marrowtransplantation and in any other process in which radiation exposureoccurs at doses greater than ordinary ambient levels, or in whichchemotherapy is used. As noted above, a significant advantage of thisinvention is that DF and/or hGH can be administered followingirradiation or chemotherapy and still achieve cytoprotection.

DF and hGH is administered prior and/or after exposure to cytotoxin. DFand hGH can be administered after chemotherapy or irradiation and stillobtain therapeutic benefit. DF and hGH have been demonstrated effectiveup to 24 hours after exposure to radiation or chemotherapy. It is likelythat each agent will be effective when administered at greater andlesser lengths of time after exposure to cytotoxin, although efficacywill vary depending upon the dose and time course of chemotherapy orirradiation, the amount, delivery route and activity of the DF and/orhGH and the condition and species of the subject. The time and frequencyof administration of the agent, like the dose and administration route,will be optimized based on clinical experience.

DF is used together with other agents conventionally employed inchemotherapy and radiotherapy, e.g. thiol therapeutics.

DF is obtained by recovery from native sources (cultures ofnon-recombinant cell lines) or is produced in recombinant cell cultureas described in EP 285,448 or Lowe et al. (supra).

EXAMPLE 1

Tissue culture fluid containing DF prepared according to Lowe et al. wasconcentrated approximately 80 fold using a Pellicon concentrator. Theconcentrated tissue culture fluid containing DF was dialyzed overnightinto 25 mM Tris-HCl, pH 8.0 at 4° C. DF was then purified using a DEAESepharose Fast Flow column equilibrated in 25 mM Tris-HCl, pH 8.0. DFwas then further purified by S-Sepharose Fast Flow. DF was eluted fromthe S-Sepharose Fast Flow column using a 0-0.4M NaCl gradient in 25 mMsuccinate, pH 4.0. Fractions containing DF were pooled, concentrated anddiafiltered into 25 mM succinate, pH 4.0, 0.1M NaCl. The concentrated DFpool was then loaded onto a Sephacryl S-300 column equilibrated in 25 mMsuccinate, pH 4.0, 0.1M NaCl. The Sephacryl S-300 fractions containingDF were pooled and dialyzed into 25 mM bis-Tris propane, pH 6.5.Endotoxin (LAL) was removed by Q-Sepharose Fast Flow. DF was dialyzedinto 25 mM succinate, pH 4.0 and stored at 4° C. at a concentration of0.39 mg/ml.

EXAMPLE 2

This example describes an improved method for DF purification.DF-containing tissue culture fluid was concentrated using either a 2liter stirred cell concentrator or a Pellicon concentrator. Theconcentrated tissue culture fluid was dialyzed overnight into 25 mMsuccinate, pH 4.0. Precipitation from the DF solution occurred in theabsence of acetonitrile or phenyl-silica. The supernatant was carefullyremoved from the precipitated proteins by either centrifugation and/ordecanting of the supernatant. The dialyzed supernatant was furtherpurified by chromatography on S-Sepharose Fast Flow. DF was eluted usinga 0-0.4M NaCl gradient in 25 mM succinate, pH 4.0. DF containingfractions (typically at about 0.1-0.3M NaCl) were pooled andconcentrated approximately 8-fold in an Amicon stirred cell. Theconcentrated DF pool was further purified on a Sephacryl S-300 columnequilibrated with 25 mM succinate, pH 4.0, 0.1M NaCl. Fractions (atabout 40-45 kD) containing D-factor were pooled and stored at 4° C.

EXAMPLE 3

This study was conducted at the U.S. Armed Forces Radiobiology Instituteby Dr. Ruth Neta. DF prepared by the method of Example 1 was formulatedin phosphate buffered saline at 1 microgram/ml. C3H/HEJ mice wereirradiated with 750 rads from a gamma radiation source. Groups of micewere injected intraperitoneally one hour after irradiation with DF dosesof either 7.5 micrograms or 1 microgram and the animals observed formortality over a period of 20 days. As can be seen from FIG. 1, all ofthe control animals had died by 17 days post-irradiation, whereas onlyabout half of the 1 microgram cohort had expired at 20 days and none ofthe animals receiving 7.5 micrograms had died.

EXAMPLE 4

This study was conducted at the Memorial Sloan Kettering Cancer Centerby Dr. Malcolm A. S. Moore. Hematopoietic protection in vivo in miceagainst high dose cyclophosphamide (Cytoxan brand) provided by a pre- orpost injection of DF was compared. Myelosuppression was monitored bydaily white blood cell counts and absolute neutrophil counts (ANC). Thedose of Cytoxan used (350 mg/kg) is lethal in 50% of mice after one doseand 90% fatal after two in the absence of supportive therapy. Daily IL-1treatment had been shown to significantly reduce the duration of theneutrophil nadir, accelerate neutrophil regeneration and enhancesurvived.

24 male Balb/c mice (Jax--average weight 23 grams) were divided into 8groups and treated as follows:

A. Control i.p. injection of 350 mg/kg Cytoxan in 0.2 ml on Day 2.

B. 5 μg DF given i.p. on Day 1, followed 24 hours later by 350 mg/kgCytoxan i.p.

C. i.p. injection of 350 mg/kg Cytoxan on Day 2 followed 6 hours laterby 5 μg DF i.p. daily for 4 days.

Data from this experiment is shown in FIG. 3. DF given pre-Cytoxan hadno significant effect (except perhaps at day 6 after Cytoxan) on eitherneutrophils or lymphocytes. DF given post-Cytoxan significantly reduced(or eliminated) the rapid drop in neutrophils and lymphocytes in thefirst 5 days post-Cytoxan and was significantly better in this regardthan IL-1, G-CSF (data not shown). DF reduced the absolute nadir of ANCbut did not accelerate regeneration.

EXAMPLE 5

This experiment was done by Dr. Adel A. Yunis at the University of MiamiSchool of Medicine. Protection in young male rats against cytosinearabinoside (ARA-C)-induced alopecia was compared for IMUVERT (abiologic response modifier derived from the bacterium Serratiamarcescens), recombinant murine TNF-α, human IL-1 beta, human DF andhGH, following the procedures described in Hussein et al., Science249:1564-1566 (28 Sep. 1990), hereby incorporated by reference.Thirty-six 8-day-old rats were randomly divided into 6 groups andtreated for a total of seven consecutive days. Each group received 20mg/kg per day ARA-C at noon on each day. Additionally and three hoursprior to the ARA-C administration, Group I received 0.1 ml buffer, groupII received 25 μg/day IMUVERT, Group III received 2.5 μg/day murineTNF-α, Group IV received 0.5 μg/day human IL-1 beta, Group V received 10μg/day DF, and Group VI received 10 μg/day hGH. Alopecia data wererecorded on day 7 of the experiment, although unpublished data confirmthe same results on day 10.

Results. Alopecia to a degree of total hair loss was reported for all 6of the control animals. The 6 rats treated with IMUVERT, one rat treatedwith TNF, one rat treated with DF, and the 6 treated with IL-1 beta hadno detectable alopecia. Four rats treated with DF had mild alopeciadefined as less than 50% hair loss and 1 DF-treated rat had moderatealopecia, defined as moderate alopecia with more than 50% hair loss. Onerat treated with hGH had mild alopecia, one rat treated with hGH hadmoderate alopecia, and 4 rats treated with hGH had severe alopecia ortotal hair loss. From this data it may be seen that TNF, IL-1, DF or hGHtreatment provides protection against chemotherapeutic alopecia.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ProLeuProIleThrProValAsnAlaThrCysAlaIleArgHis                                 151015                                                                        ProCysHisAsnAsnLeuMetAsnGlnIleArgSerGlnLeuAla                                 202530                                                                        GlnLeuAsnGlySerAlaAsnAlaLeuPheIleLeuTyrTyrThr                                 354045                                                                        AlaGlnGlyGluProPheProAsnAsnLeuAspLysLeuCysGly                                 505560                                                                        ProAsnValThrAspPheProProPheHisAlaAsnGlyThrGlu                                 657075                                                                        LysAlaLysLeuValGluLeuTyrArgIleValValTyrLeuGly                                 808590                                                                        ThrSerLeuGlyAsnIleThrArgAspGlnLysIleLeuAsnPro                                 95100105                                                                      SerAlaLeuSerLeuHisSerLysLeuAsnAlaThrAlaAspIle                                 110115120                                                                     LeuArgGlyLeuLeuSerAsnValLeuCysArgLeuCysSerLys                                 125130135                                                                     TyrHisValGlyHisValAspValThrTyrGlyProAspThrSer                                 140145150                                                                     GlyLysAspValPheGlnLysLysLysLeuGlyCysGlnLeuLeu                                 155160165                                                                     GlyLysTyrLysGlnIleIleAlaValLeuAlaGlnAlaPhe                                    170175179                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProLeuProIleThrProValAsnAlaThrCysAlaIleArgHis                                 151015                                                                        ProCysHisGlyAsnLeuMetAsnGlnIleLysAsnGlnLeuAla                                 202530                                                                        GlnLeuAsnGlySerAlaAsnAlaLeuPheIleSerTyrTyrThr                                 354045                                                                        AlaGlnGlyGluProPheProAsnAsnValGluLysLeuCysAla                                 505560                                                                        ProAsnMetThrAspPheProSerPheHisGlyAsnGlyThrGlu                                 657075                                                                        LysThrLysLeuValGluLeuTyrArgMetValAlaTyrLeuSer                                 808590                                                                        AlaSerLeuThrAsnIleThrArgAspGlnLysValLeuAsnPro                                 95100105                                                                      ThrAlaValSerLeuGlnValLysLeuAsnAlaThrIleAspVal                                 110115120                                                                     MetArgGlyLeuLeuSerAsnValLeuCysArgLeuCysAsnLys                                 125130135                                                                     TyrArgValGlyHisValAspValProProValProAspHisSer                                 140145150                                                                     AspLysGluAlaPheGlnArgLysLysLeuGlyCysGlnLeuLeu                                 155160165                                                                     GlyThrTyrLysGlnValIleSerValValValGlnAlaPhe                                    170175179                                                                     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We claim:
 1. A method for treating injury resulting from radiation orchemotherapy comprising:administering to a subject a synergisticcytoprotective dose of at least two members selected from the groupconsisting of D-factor, growth hormone (GH), tumor necrosis factor (TNF)and IL-1 to the subject, provided that where TNF and IL-1 are bothadministered a third member is also administered, wherein thecytoprotective dose is administered prior to, during, or after thesubject is exposed to radiation or chemotherapy.
 2. The method of claim1 wherein the subject has myeloid leukemia.
 3. The method of claim 1wherein the subject has a non-leukemic tumor.
 4. The method of claim 1wherein the subject undergoes a bone marrow transplant followingirradiation or chemotherapy.
 5. The method of claim 1 wherein D-factoror GH is administered after exposure to a radiation or chemotherapyinjury.
 6. The method of claim 1 wherein TNF or IL-1 is administeredprior to or concurrent with exposure to a radiation or chemotherapyinjury.
 7. The method of claim 1 wherein the TNF is TNF-α.